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Abstract Background There is a higher risk for nematode infections associated with outdoor-reared pigs. Next to Ascaris suum, Oesophagostomum dentatum and Trichuris suis, there is the potential of infections with other nodular worm species, Hyostrongylus rubidus, Stongyloides ransomi and Metastrongylus spp. lungworms. Next-generation sequencing methods describing the nemabiome have not yet been established for porcine nematodes. Methods FLOTAC was used for faecal egg counts of porcine gastrointestinal nematodes and lungworms in piglets, fatteners and adults individually. A nemabiome analyses based on ITS-2 gene region metabarcoding was used to differentiate strongyle species. Additionally, questionnaire data was analysed using mixed-effect regression to identify potential risk factors associated with parasite occurrences and egg shedding intensity. Results On 15 of 17 farms nematode eggs were detected. Ascaris suum, strongyles and T. suis were detected on 82%, 70% and 35% of the 17 farms, respectively. Lungworms were detected on one out of four farms with access to pasture. Strongyloides ransomi was not detected. 32% (CI 28–36%), 27% (24–31%), 5% (4–7%) and 3% (0.9–8%) of the samples where tested positive for strongyles, A. suum, T. suis and lungworms, respectively. The nemabiome analysis revealed three different strongyle species, with O. dentatum being the most common (mean 93.9%), followed by O. quadrispinulatum (5.9%) and the hookworm Globocephalus urosubulatus (0.1%). The bivariate and multivariate risk factor analyses showed among others that cleaning once a week compared to twice a week increased the odds significantly for being infected with A. suum (OR 78.60) and strongyles (2077.59). Access to pasture was associated with higher odds for A. suum (43.83) and strongyles (14.21). Compared to shallow litter systems, deep litter and free range systems resulted in significant higher odds for strongyles (85.74, 215.59, respectively) and T. suis (200.33, 623.08). Conclusions Infections with A. suum, O. dentatum, O. quadrispinulatum, T. suis, Metastrongylus spp. and G. urosubulatus are present in German outdoor-reared pigs. This is the first report of G. urosubulatus in domestic pigs in Europe. Metabarcoding based on the ITS-2 region is a suitable tool to analyse the porcine nemabiome. Furthermore, management practices have the potential of reducing the risk of parasite infections.

Background: There is a higher risk for nematode infections associated with outdoor-reared pigs. Next to Ascaris suum, Oesophagostomum dentatum and Trichuris suis, there is the potential of infections with other nodular worm species, Hyostrongylus rubidus, Stongyloides ransomi and Metastrongylus spp. lungworms. Next-generation sequencing methods describing the nemabiome have not yet been established for porcine nematodes. Methods: FLOTAC was used for faecal egg counts of porcine gastrointestinal nematodes and lungworms in piglets, fatteners and adults individually. A nemabiome analyses based on ITS-2 gene region metabarcoding was used to differentiate strongyle species. Additionally, questionnaire data was analysed using mixed-effect regression to identify potential risk factors associated with parasite occurrences and egg shedding intensity. Results: On 15 of 17 farms nematode eggs were detected. Ascaris suum, strongyles and T. suis were detected on 82%, 70% and 35% of the 17 farms, respectively. Lungworms were detected on one out of four farms with access to pasture. Strongyloides ransomi was not detected. 32% (CI 28–36%), 27% (24–31%), 5% (4–7%) and 3% (0.9–8%) of the samples where tested positive for strongyles, A. suum, T. suis and lungworms, respectively. The nemabiome analysis revealed three different strongyle species, with O. dentatum being the most common (mean 93.9%), followed by O. quadrispinulatum (5.9%) and the hookworm Globocephalus urosubulatus (0.1%). The bivariate and multivariate risk factor analyses showed among others that cleaning once a week compared to twice a week increased the odds significantly for being infected with A. suum (OR 78.60) and strongyles (2077.59). Access to pasture was associated with higher odds for A. suum (43.83) and strongyles (14.21). Compared to shallow litter systems, deep litter and free range systems resulted in significant higher odds for strongyles (85.74, 215.59, respectively) and T. suis (200.33, 623.08). Conclusions: Infections with A. suum, O. dentatum, O. quadrispinulatum, T. suis, Metastrongylus spp. and G. urosubulatus are present in German outdoor-reared pigs. This is the first report of G. urosubulatus in domestic pigs in Europe. Metabarcoding based on the ITS-2 region is a suitable tool to analyse the porcine nemabiome. Furthermore, management practices have the potential of reducing the risk of parasite infections. [ABSTRACT FROM AUTHOR]

Anthelmintic resistance occurs worldwide in strongyles of ruminants but data from low-income countries are sparse and rarely apply most up to date methods, while effects of management practices in these countries are poorly documented. In Mozambique, benzimidazole resistance has been previously reported; the present study followed this up in detail, applying in vivo faecal egg count (FEC) reduction test (FECRT), in vitro egg hatch test (EHT) and molecular deep amplicon sequencing approaches targeting the internal transcribed spacer 2 (ITS-2, nemabiome) and the isotype 1 β-tubulin gene to determine the resistance status on farms and the strongyle species involved. Adult Landim goats (433) from six semi-intensive and ten extensive farms (22–30 animals/farm) from Maputo Province were visited April 2021 to February 2022. Fenbendazole (5 mg/kg bw, Panacur®) was administered orally and FEC determined using Mini-FLOTAC. The eggCounts package was used to calculate FECRs with 90% confidence intervals from paired day 0 and 14 data. In vivo and in vitro tests detected AR on 5/16 (31%) farms. This included 1/10 extensive and 4/6 semi-intensive farms. The odds of finding resistant strongyles on a semi-intensive commercial farm was 40-fold higher than on an extensive farm (p = 0.016, logistic regression). A strong, negative correlation was observed between FECRT and EHT EC50 values (Pearson's R = −0.83, P = 0.001; Cohen's κ coefficient 1.0). Nemabiome data showed that Haemonchus contortus, Trichostrongylus colubriformis and unclassified Oesophagostomum closely related to Oesophagostomum columbianum were most abundant before treatment and in particular H. contortus frequencies increased after treatment. Benzimidazole resistance associated polymorphisms were detected in H. contortus and T. colubriformis. Moreover, there were hints that resistance alleles were present in Trichostrongylus axei and Teladorsagia circumcincta. Farmers should regularly test the efficacy of anthelmintics used and consider more sustainable worm control approaches to reduce selection for resistance.

The ITS‐2‐rRNA has been particularly useful for nematode metabarcoding but does not resolve all phylogenetic relationships, and reference sequences are not available for many nematode species. This is a particular issue when metabarcoding complex communities such as wildlife parasites or terrestrial and aquatic free‐living nematode communities. We have used markerDB to produce four databases of distinct regions of the rRNA cistron: the 18S rRNA gene, the 28S rRNA gene, the ITS‐1 intergenic spacer and the region spanning ITS‐1_5.8S_ITS‐2. These databases comprise 2645, 254, 13,461 and 10,107 unique full‐length sequences representing 1391, 204, 1837 and 1322 nematode species, respectively. The comparative analysis illustrates the complementary value but also reveals a better representation of Clade III, IV and V than Clade I and Clade II nematodes in each case. Although the ITS‐1 database includes the largest number of unique full‐length sequences, the 18S rRNA database provides the widest taxonomic coverage. We also developed PrimerTC, a tool to assess primer sequence conservation across any reference sequence database, and have applied it to evaluate a large number of previously published rRNA cistron primers. We identified sets of primers that currently provide the broadest taxonomic coverage for each rRNA marker across the nematode phylum. These new resources will facilitate more comprehensive metabarcoding of nematode communities using either short‐read or long‐read sequencing platforms. Further, PrimerTC is available as a simple WebApp to guide or assess PCR primer design for any genetic marker and/or taxonomic group beyond the nematode phylum. [ABSTRACT FROM AUTHOR]

Abstract Background Mixed strongylid infections significantly impact equine health and performance. Traditional microscopy-based methods exhibit limitations in accurately identifying strongylid species. Nemabiome deep amplicon sequencing approach previously succeeded in describing the strongylid communities in livestock including equids. However, there are no available studies that describe the structural communities of strongylid parasites in horses in Thailand. Therefore, this study was undertaken encompassing the ITS-2 rDNA metabarcoding assay to characterize strongylid species within horse fecal samples collected from a cohort of yearlings at the largest domesticated stud farm in Thailand. In addition, to investigate the capability of ITS-2 rDNA in assessing the phylogenetic relationships among the identified strongylid species. Results The study identified 14 strongylid species in the examined equine populations, each with varying prevalence. Notably, Cylicocyclus nassatus and Cylicostephanus longibursatus were identified as the predominant species, with Strongylus spp. conspicuously absent. The phylogenetic analysis of 207 amplicon sequence variants (ASVs) displayed a complex relationship among the investigated cyathostomin species, with some species are positioned across multiple clades, demonstrating close associations with various species and genera. Conclusion The ITS-2 nemabiome sequencing technique provided a detailed picture of horse strongylid parasite species in the studied population. This establishes a foundation for future investigations into the resistance status of these parasites and enables efforts to mitigate their impact.

[Display omitted] • Deep amplicon sequencing of levamisole (LEV)-resistant S168T in Haemonchus spp. from Australia was undertaken. • A new approach was developed, enabling nemabiome and multi-drug surveillance in field populations. • Mixed amplicon sequencing was equivalent to individual amplicon sequencing. • LEV-resistant S168T was present in most ovine and caprine Australian field isolates. Anthelmintic-resistant parasitic nematodes present a significant threat to sustainable livestock production worldwide. The ability to detect the emergence of anthelmintic resistance at an early stage, and therefore determine which drugs remain most effective, is crucial for minimising production losses. Despite many years of research into the molecular basis of anthelmintic resistance, no molecular-based tools are commercially available for the diagnosis of resistance as it emerges in field settings. We describe a mixed deep amplicon sequencing approach to determine the frequency of the levamisole (LEV)-resistant single nucleotide polymorphism (SNP) within arc -8 exon 4 (S168T) in Haemonchus spp., coupled with benzimidazole (BZ)-resistant SNPs within β -tubulin isotype-1 and the internal transcribed spacer-2 (ITS-2) nemabiome. This constitutes the first known multi-drug and multi-species molecular diagnostic developed for helminths of veterinary importance. Of the ovine, bovine, caprine and camelid Australian field isolates we tested, S168T was detected in the majority of Haemonchus spp. populations from sheep and goats, but rarely at a frequency greater than 16%; an arbitrary threshold we set based on whole genome sequencing (WGS) of LEV-resistant Haemonchus contortus GWBII. Overall, BZ resistance was far more prevalent in Haemonchus spp. than LEV resistance, confirming that LEV is still an effective anthelmintic class for small ruminants in New South Wales, Australia. The mixed amplicon metabarcoding approach described herein paves the way towards the use of large scale sequencing as a surveillance technology in the field, the results of which can be translated into evidence-based recommendations for the livestock sector. [ABSTRACT FROM AUTHOR]

Abstract Background Veterinary diagnostics aid intervention strategies, track zoonoses, and direct selective breeding programs in livestock. In ruminants, gastrointestinal nematode (GIN) parasites are a major cause of production losses, but morphologically similar species limit our understanding of how specific GIN co-infections impact health in resource-limited settings. To estimate the presence and relative abundance of GINs and other helminths at the species level, we sought to develop a low-cost and low-resource molecular toolkit applied to goats from rural Malawi smallholdings. Methods Goats were subjected to health scoring and faecal sampling on smallholdings in Lilongwe district, Malawi. Infection intensities were estimated by faecal nematode egg counts with a faecal subsample desiccated for DNA analysis. Two DNA extraction methods were tested (low-resource magbead kit vs high-resource spin-column kit), with resulting DNA screened by endpoint polymerase chain reaction (PCR), semi-quantitative PCR, quantitative PCR (qPCR), high-resolution melt curve analysis (HRMC), and ‘nemabiome’ internal transcribed spacer 2 (ITS-2) amplicon sequencing. Results Both DNA isolation methods yielded comparable results despite poorer DNA purity and faecal contaminant carryover from the low-resource magbead method. GINs were detected in 100% of samples regardless of infection intensity. Co-infections with GINs and coccidia (Eimeria spp.) were present in most goats, with GIN populations dominated by Haemonchus contortus, Trichostrongylus colubriformis, Trichostrongylus axei, and Oesophagostomum columbianum. Both multiplex PCR and qPCR were highly predictive of GIN species proportions obtained using nemabiome amplicon sequencing; however, HRMC was less reliable than PCR in predicting the presence of particular species. Conclusions These data represent the first ‘nemabiome’ sequencing of GINs from naturally infected smallholder goats in Africa and show the variable nature of GIN co-infections between individual animals. A similar level of granularity was detected by semi-quantitative PCR methods, which provided an accurate summary of species composition. Assessing GIN co-infections is therefore possible using cost-efficient low-resource DNA extraction and PCR approaches that can increase the capacity of molecular resources in areas where sequencing platforms are not available; and also open the door to affordable molecular GIN diagnostics. Given the diverse nature of infections in livestock and wildlife, these approaches have potential for disease surveillance in other areas. Graphical Abstract

Hookworms (Ancylostomatidae) are well‐known parasites in dogs due to their health impacts and zoonotic potential. While faecal analysis is the traditional method for detection, improvements in husbandry and deworming have decreased their prevalence in urban owned dogs. Drug resistance in Ancylostoma caninum is becoming a discussion point in small animal practices across the region. This study aimed to identify hookworm species present in Australian and New Zealand dogs using molecular techniques. The ITS‐2 and isotype‐1 β‐tubulin assays were used to identify and quantify hookworm species. Results showed absence of coinfection in Australian samples from Greater Sydney region belonging either to A. caninum or Uncinaria stenocephala, while New Zealand samples were a mixture of A. caninum and U. stenocephala. The amplified isotype‐1 β‐tubulin sequences exhibited susceptibility to benzimidazole drugs. Rare mutations were identified in A. caninum and U. stenocephala sequences, representing a small percentage of reads. This study highlights the importance of molecular techniques in accurately identifying and quantifying hookworm species in dog populations. [ABSTRACT FROM AUTHOR]

Gastrointestinal nematode parasites are of major concern for horses, where Strongylus vulgaris is considered the most pathogenic among the Strongylus species. Diagnosis of S. vulgaris infections can be determined with next generation sequencing techniques, which are inherently dependent on reference sequences. The best marker for parasitic nematodes is internal transcribed spacer 2 (ITS2) and we provide the first complete ITS2 sequences from five morphologically identified S. vulgaris and additional sequences from two S. edentatus. These sequences have high similarity to already published partial sequences and amplicon sequence variants (ASV) based on next generation sequencing (NGS). The ITS2 sequences from S. vulgaris matched available partial ITS2 sequences and the full ASVs, whereas the S. edentatus sequence matched another complete sequence. We also compare Sanger sequencing and NGS methods and conclude that the ITS2 variation is better represented with NGS methods. Based on this, we recommend that further sequencing of morphologically identified specimens of various species should be performed with NGS cover the intraspecific variation in the ITS2. [ABSTRACT FROM AUTHOR]

Abstract Background Although wild ungulate populations are heavily monitored throughout Europe, we understand little of how parasites affect population dynamics, and there is no systematic, long-term monitoring of parasite diversity and parasite loads. Such monitoring is in part hampered by a lack of time- and cost-effective assay methodologies with high sensitivity and good taxonomic resolution. DNA metabarcoding has been successfully used to characterize the parasitic nemabiome with high taxonomic resolution in a variety of wild and domestic hosts. However, in order to implement this technique in large-scale, potentially non-invasive monitoring of gastrointestinal parasitic nematodes (GIN), protocol optimization is required to maximize biodiversity detection, whilst maintaining time- and cost-effectiveness. Methods Faecal samples were collected from a wild moose population and GIN communities were characterized and quantified using both parasitological techniques (egg and larva counting) and DNA metabarcoding of the ITS2 region of rDNA. Three different isolation methods were compared that differed in the volume of starting material and cell lysis method. Results Similar nematode faunas were recovered from all samples using both parasitological and metabarcoding methods, and the approaches were largely congruent. However, metabarcoding assays showed better taxonomic resolution and slightly higher sensitivity than egg and larvae counts. The metabarcoding was not strictly quantitative, but the proportion of target nematode sequences recovered was correlated with the parasitologically determined parasite load. Species detection rates in the metabarcoding assays were maximized using a DNA isolation method that included mechanical cell disruption and maximized the starting material volume. Conclusions DNA metabarcoding is a promising technique for the non-invasive, large-scale monitoring of parasitic GINs in wild ungulate populations, owing to its high taxonomic resolution, increased assay sensitivity, and time- and cost-effectiveness. Although metabarcoding is not a strictly quantitative method, it may nonetheless be possible to create a management- and conservation-relevant index for the host parasite load from this data. To optimize the detection rates and time- and cost-effectiveness of metabarcoding assays, we recommend choosing a DNA isolation method that involves mechanical cell disruption and maximizes the starting material volume. Graphical Abstract

Background: Veterinary diagnostics aid intervention strategies, track zoonoses, and direct selective breeding programs in livestock. In ruminants, gastrointestinal nematode (GIN) parasites are a major cause of production losses, but morphologically similar species limit our understanding of how specific GIN co-infections impact health in resource-limited settings. To estimate the presence and relative abundance of GINs and other helminths at the species level, we sought to develop a low-cost and low-resource molecular toolkit applied to goats from rural Malawi smallholdings. Methods: Goats were subjected to health scoring and faecal sampling on smallholdings in Lilongwe district, Malawi. Infection intensities were estimated by faecal nematode egg counts with a faecal subsample desiccated for DNA analysis. Two DNA extraction methods were tested (low-resource magbead kit vs high-resource spin-column kit), with resulting DNA screened by endpoint polymerase chain reaction (PCR), semi-quantitative PCR, quantitative PCR (qPCR), high-resolution melt curve analysis (HRMC), and 'nemabiome' internal transcribed spacer 2 (ITS-2) amplicon sequencing. Results: Both DNA isolation methods yielded comparable results despite poorer DNA purity and faecal contaminant carryover from the low-resource magbead method. GINs were detected in 100% of samples regardless of infection intensity. Co-infections with GINs and coccidia (Eimeria spp.) were present in most goats, with GIN populations dominated by Haemonchus contortus, Trichostrongylus colubriformis, Trichostrongylus axei, and Oesophagostomum columbianum. Both multiplex PCR and qPCR were highly predictive of GIN species proportions obtained using nemabiome amplicon sequencing; however, HRMC was less reliable than PCR in predicting the presence of particular species. Conclusions: These data represent the first 'nemabiome' sequencing of GINs from naturally infected smallholder goats in Africa and show the variable nature of GIN co-infections between individual animals. A similar level of granularity was detected by semi-quantitative PCR methods, which provided an accurate summary of species composition. Assessing GIN co-infections is therefore possible using cost-efficient low-resource DNA extraction and PCR approaches that can increase the capacity of molecular resources in areas where sequencing platforms are not available; and also open the door to affordable molecular GIN diagnostics. Given the diverse nature of infections in livestock and wildlife, these approaches have potential for disease surveillance in other areas. [ABSTRACT FROM AUTHOR]

Abstract Background Wildlife hosts may serve as reservoirs for strongyles, which can be transmitted to domestic livestock. Therefore, studies evaluating nemabiome compositions in wildlife ruminants are of great use in assessing the possibility of transmission of important nematode pathogens to domestic sheep in Sweden. Methods First, fecal samples were collected from roe deer (n = 125), fallow deer (n = 106), red deer (n = 18) and mouflon (n = 13) in south central Sweden during the hunting season in 2019. Second, after fecal examination samples were cultured and the larvae were harvested, followed by DNA extractions. Third, all samples were barcoded and processed for sequence analysis on the PacBio platform. Finally, bioinformatic sequence analysis was conducted with DADA2, while species diversity and richness, as well as interactions between the different hosts, were calculated and analyzed in R. Results Nematode ITS2 sequences were found in 225 of 262 (86%) samples. In total, 31 taxa were identified, among which 26 (86%) to the species level. These were found in different combinations, among which 24 (77%) occurred in roe deer, 19 (61%) in fallow deer, 20 (65%) in red deer and 10 (32%) in mouflon. Five of the species found are known to be associated with livestock (Chabertia ovina, Haemonchus contortus, Oesophagostomum venulosum, Teladorsagia circumcincta and Trichostrongylus axei). However, in the present study the relative abundance and prevalence of most of these species were low. The most striking exception was T. axei, which was relatively abundant in all wildlife hosts. Mostly a wide range of wildlife specific nematodes such as Ostertagia leptospicularis and Spiculopteragia spp. were identified including the invasive nematode Spiculopteragia houdemeri, which was found for the first time in red deer, fallow deer, and mouflon in Sweden. The difference in the number of shared species between mouflon and all cervids (n = 6) was less than among all three cervids (n = 8). Conclusion In this study, we investigated the community structure of parasitic intestinal nematodes in four wildlife hosts, and we found that the majority of the parasite species identified were wildlife specific. We also found a new, potentially invasive species not reported before. After comparing the nemabiome of the wildlife hosts in this study with a previous study in sheep from the same geographical region, we conclude that the horizontal transmission potential appears to be relatively low. Still, cross-infections of nematodes between game and sheep cannot be completely ignored. Graphical Abstract

Abstract Background Next-generation sequencing (NGS) has provided an alternative strategy to study the composition of nematode communities with increased resolution and sensitivity. However, the handling and processing of gigabytes worth of amplicon sequence data produced by an NGS platform is still a major hurdle, limiting the use and adoption of faster and more convenient analysis software. Methods In total 32 paired, fecal samples from Swedish sheep flocks were cultured and the larvae subsequently harvested subjected to internal transcribed spacer 2 (ITS2) amplicon sequencing using the PacBio platform. Samples were analyzed with three different bioinformatic pipelines, i.e. the DADA2, Mothur and SCATA pipelines, to determine species composition and richness. Results For the the major species tested in this study (Haemonchus contortus, Teladorsagia circumcinta and Trichostrongylus colubriformis) neither relative abundances nor species diversity differed significantly between the three pipelines, effectively showing that all three analysis pipelines, although different in their approaches, yield nearly identical outcomes. In addition, the samples analyzed here had especially high frequencies of H. contortus (90–95% across the three pipelines) both before and after sample treatment, followed by T. circumcinta (3.5–4%). This shows that H. contortus is the parasite of primary importance in contemporary Swedish sheep farms struggling with anthelmintic resistance. Finally, although on average a significant reduction in egg counts was achieved post-treatment, no significant shifts in major species relative frequencies occurred, indicating highly rigid community structures at sheep farms where anthelmintic resistance has been reported. Conclusions The findings presented here further contribute to the development and application of NGS technology to study nemabiome compositions in sheep, in addition to expanding our understanding about the most recent changes in parasite species abundances from Swedish sheep farms struggling with anthelmintic resistance. Graphical Abstract

Background: Although wild ungulate populations are heavily monitored throughout Europe, we understand little of how parasites affect population dynamics, and there is no systematic, long-term monitoring of parasite diversity and parasite loads. Such monitoring is in part hampered by a lack of time- and cost-effective assay methodologies with high sensitivity and good taxonomic resolution. DNA metabarcoding has been successfully used to characterize the parasitic nemabiome with high taxonomic resolution in a variety of wild and domestic hosts. However, in order to implement this technique in large-scale, potentially non-invasive monitoring of gastrointestinal parasitic nematodes (GIN), protocol optimization is required to maximize biodiversity detection, whilst maintaining time- and cost-effectiveness. Methods: Faecal samples were collected from a wild moose population and GIN communities were characterized and quantified using both parasitological techniques (egg and larva counting) and DNA metabarcoding of the ITS2 region of rDNA. Three different isolation methods were compared that differed in the volume of starting material and cell lysis method. Results: Similar nematode faunas were recovered from all samples using both parasitological and metabarcoding methods, and the approaches were largely congruent. However, metabarcoding assays showed better taxonomic resolution and slightly higher sensitivity than egg and larvae counts. The metabarcoding was not strictly quantitative, but the proportion of target nematode sequences recovered was correlated with the parasitologically determined parasite load. Species detection rates in the metabarcoding assays were maximized using a DNA isolation method that included mechanical cell disruption and maximized the starting material volume. Conclusions: DNA metabarcoding is a promising technique for the non-invasive, large-scale monitoring of parasitic GINs in wild ungulate populations, owing to its high taxonomic resolution, increased assay sensitivity, and time- and cost-effectiveness. Although metabarcoding is not a strictly quantitative method, it may nonetheless be possible to create a management- and conservation-relevant index for the host parasite load from this data. To optimize the detection rates and time- and cost-effectiveness of metabarcoding assays, we recommend choosing a DNA isolation method that involves mechanical cell disruption and maximizes the starting material volume. [ABSTRACT FROM AUTHOR]

[Display omitted] • We studied the parasite community and population structures from sheep and roe deer. • Generalist parasite species dominated the nemabiomes of sheep but also roe deer. • The presence of livestock may have modified drastically the nemabiome of roe deer. • There was high circulation of nematodes among sheep and roe deer populations at a regional scale. The growth of livestock farming and the recent expansion of wild ungulate populations in Europe favor opportunities for direct and/or indirect cross-transmission of pathogens. Comparatively few studies have investigated the epidemiology of gastro-intestinal nematode parasites, an ubiquitous and important community of parasites of ungulates, at the wildlife/livestock interface. In this study, we aimed to assess the influence of livestock proximity on the gastrointestinal nematode community of roe deer in a rural landscape located in southern France. Using ITS-2 rDNA nemabiome metabarcoding on fecal larvae, we analysed the gastrointestinal nematode communities of roe deer and sheep. In addition, we investigated Haemonchus contortus nad4 mtDNA diversity to specifically test parasite circulation among domestic and wild host populations. The dominant gastrointestinal nematode species found in both the roe deer and sheep were generalist species commonly found in small ruminant livestock (e.g. H. contortus), whereas the more specialised wild cervid nematode species (e.g. Ostertagia leptospicularis) were only present at low frequencies. This is in marked contrast with previous studies that found the nemabiomes of wild cervid populations to be dominated by cervid specialist nematode species. In addition, the lack of genetic structure of the nad4 mtDNA of H. contortus populations between host species suggests circulation of gastrointestinal nematodes between roe deer and sheep. The risk of contact with livestock only has a small influence on the nemabiome of roe deer, suggesting the parasite population of roe deer has been displaced by generalist livestock parasites due to many decades of sheep farming, not only for deer grazing close to pastures, but also at a larger regional scale. We also observed some seasonal variation in the nemabiome composition of roe deer. Overall, our results demonstrate significant exchange of gastrointestinal nematodes between domestic and wild ungulates, with generalist species spilling over from domestic ungulates dominating wild cervid parasite communities. [ABSTRACT FROM AUTHOR]

Gastrointestinal tract (GIT) nematode infections have a significant negative impact on the well-being and productivity of animals. While it is common for a host to be co-infected with multiple species of nematode parasites simultaneously, there is a lack of effective tools to study the composition of these complex parasite communities. We describe the application of the "nemabiome" amplicon sequencing to study parasitic GIT nematode communities in captive wildlife at the National Zoological Garden, South African National Biodiversity Institute. A total of 13 samples from Roan antelope (n=1), Eland (n=1), Sable antelope (n=9), Arabian oryx (n=1), and Blue duiker (n=1) were analysed for the presence of GIT nematode infections. A total of 256 operational taxonomical units (OTUs) were generated and compared to a curated database with ≥99% identity to sequences in the NCBI database. The OTUs found represented nematode species which are commonly associated with wild ruminants. These belonged to members of the genera Haemonchus, Trichostrongylus , and Cooperia , with a few OTUs classified as unidentified genera detected. These were further classified to species level, with Haemonchus contortus and Trichostrongylus colubriformis having high relative abundance across all captive ungulate species. Utilizing amplicon sequencing to examine the nemabiome holds great potential for enhancing our basic knowledge of the biology of gastrointestinal nematodes and monitoring infections. This improved understanding could guide the development of more efficacious preventive and control measures against these significant parasites, which negatively impact animal health and productivity on a global scale.

Cyathostomins are the most abundant equid endoparasites globally. There are approximately fifty cyathostomin species and, whilst they occupy distinct niches within the large intestine, they are generally considered to share similar characteristics in terms of pathogenicity and response to drug treatment. There are three classes of anthelmintic licensed in the UK to treat cyathostomins (benzimidazoles, tetrahydropyrimidines and macrocyclic lactones) and cases of resistance have been documented for all classes. Previously, faecal egg count reduction tests (FECRT) on four UK Thoroughbred studs revealed multidrug resistant cyathostomins on one stud (A), with evidence of resistance to the macrocyclic lactones (MLs) ivermectin (IVM) and moxidectin (MOX), and to pyrantel (PYR). The remaining three studs (B-D) lacked resistance to IVM and MOX but had a shortened egg reappearance period post treatment. To determine whether specific species could be associated with the observed resistance and shortened egg reappearance period, strongyle eggs collected from between six and 15 individual horses per stud were copro-cultured to third larval stage (L3), before and after anthelmintic treatment, over a three-year timeframe (2021-2023). Quantitative DNA metabarcoding of the ITS-2 region was carried out on all samples. On stud A, single but differing species were found to be responsible for ML and pyrantel resistance in yearlings, Cyathostomum catinatum and Cylicocyclus nassatus, respectively. On studs B-D, with shortened egg reappearance periods, species composition remained largely unchanged post treatment. This study is the first to quantitatively profile cyathostomin species composition pre- and post-treatment in a multidrug resistant population in the UK, revealing that resistance in cyathostomins was species specific. This raises the question of whether these species may be responsible for ML and PYR resistance more widely and indicates that anthelmintic resistance in cyathostomins may not be a multi-species phenomenon., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Identifying factors that drive among-individual variation in mixed parasitic infections is fundamental to understanding the ecology and evolution of host–parasite interactions. However, a lack of non-invasive diagnostic tools to quantify mixed infections has restricted their investigation for host populations in the wild. This study applied DNA metabarcoding on parasite larvae cultured from faecal samples to characterize mixed strongyle infections of 320 feral horses on Sable Island, Nova Scotia, Canada, in 2014 to test for the influence of host (age, sex and reproductive/social status) and environmental (location, local density and social group membership) factors on variation. Twenty-five strongyle species were identified, with individual infections ranging from 3 to 18 species with a mean richness (±1 s.d.) of 10.8 ± 3.1. Strongyle eggs shed in faeces were dominated by small strongyle (cyathostomins) species in young individuals, transitioning to large strongyles ( Strongylus spp.) in adults. Egg counts were highest in young individuals and in the west or centre of the island for most species. Individuals in the same social group had similar parasite communities, supporting the hypothesis that shared environment may drive parasite assemblages. Other factors such as local horse density, sex, date and reproductive/social status had minimal impacts on infection patterns. This study demonstrates that mixed infections can be dynamic across host ontogeny and space and emphasizes the need to consider species-specific infection patterns when investigating mixed infections.

Anthelmintic resistance occurs worldwide in strongyles of ruminants but data from low-income countries are sparse and rarely apply most up to date methods, while effects of management practices in these countries are poorly documented. In Mozambique, benzimidazole resistance has been previously reported; the present study followed this up in detail, applying in vivo faecal egg count (FEC) reduction test (FECRT), in vitro egg hatch test (EHT) and molecular deep amplicon sequencing approaches targeting the internal transcribed spacer 2 (ITS-2, nemabiome) and the isotype 1 β-tubulin gene to determine the resistance status on farms and the strongyle species involved. Adult Landim goats (433) from six semi-intensive and ten extensive farms (22-30 animals/farm) from Maputo Province were visited April 2021 to February 2022. Fenbendazole (5 mg/kg bw, Panacur®) was administered orally and FEC determined using Mini-FLOTAC. The eggCounts package was used to calculate FECRs with 90% confidence intervals from paired day 0 and 14 data. In vivo and in vitro tests detected AR on 5/16 (31%) farms. This included 1/10 extensive and 4/6 semi-intensive farms. The odds of finding resistant strongyles on a semi-intensive commercial farm was 40-fold higher than on an extensive farm (p = 0.016, logistic regression). A strong, negative correlation was observed between FECRT and EHT EC 50 values (Pearson's R = -0.83, P = 0.001; Cohen's κ coefficient 1.0). Nemabiome data showed that Haemonchus contortus, Trichostrongylus colubriformis and unclassified Oesophagostomum closely related to Oesophagostomum columbianum were most abundant before treatment and in particular H. contortus frequencies increased after treatment. Benzimidazole resistance associated polymorphisms were detected in H. contortus and T. colubriformis. Moreover, there were hints that resistance alleles were present in Trichostrongylus axei and Teladorsagia circumcincta. Farmers should regularly test the efficacy of anthelmintics used and consider more sustainable worm control approaches to reduce selection for resistance., Competing Interests: Declaration of competing interest Georg von Samson-Himmelstjerna is a member of the editorial board of Int. J. Parasitol. Drugs Drug Rest. Furthermore, he declares that he has previous and ongoing research and consultancy collaborations with several veterinary pharmaceutical and diagnostic companies. All other authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Wildlife hosts may serve as reservoirs for strongyles, which can be transmitted to domestic livestock. Therefore, studies evaluating nemabiome compositions in wildlife ruminants are of great use in assessing the possibility of transmission of important nematode pathogens to domestic sheep in Sweden. Methods: First, fecal samples were collected from roe deer (n = 125), fallow deer (n = 106), red deer (n = 18) and mouflon (n = 13) in south central Sweden during the hunting season in 2019. Second, after fecal examination samples were cultured and the larvae were harvested, followed by DNA extractions. Third, all samples were barcoded and processed for sequence analysis on the PacBio platform. Finally, bioinformatic sequence analysis was conducted with DADA2, while species diversity and richness, as well as interactions between the different hosts, were calculated and analyzed in R. Results: Nematode ITS2 sequences were found in 225 of 262 (86%) samples. In total, 31 taxa were identified, among which 26 (86%) to the species level. These were found in different combinations, among which 24 (77%) occurred in roe deer, 19 (61%) in fallow deer, 20 (65%) in red deer and 10 (32%) in mouflon. Five of the species found are known to be associated with livestock (Chabertia ovina, Haemonchus contortus, Oesophagostomum venulosum, Teladorsagia circumcincta and Trichostrongylus axei). However, in the present study the relative abundance and prevalence of most of these species were low. The most striking exception was T. axei, which was relatively abundant in all wildlife hosts. Mostly a wide range of wildlife specific nematodes such as Ostertagia leptospicularis and Spiculopteragia spp. were identified including the invasive nematode Spiculopteragia houdemeri, which was found for the first time in red deer, fallow deer, and mouflon in Sweden. The difference in the number of shared species between mouflon and all cervids (n = 6) was less than among all three cervids (n = 8). Conclusion: In this study, we investigated the community structure of parasitic intestinal nematodes in four wildlife hosts, and we found that the majority of the parasite species identified were wildlife specific. We also found a new, potentially invasive species not reported before. After comparing the nemabiome of the wildlife hosts in this study with a previous study in sheep from the same geographical region, we conclude that the horizontal transmission potential appears to be relatively low. Still, cross-infections of nematodes between game and sheep cannot be completely ignored. [ABSTRACT FROM AUTHOR]

Alternative strategies to chemical anthelmintics are needed for the sustainable control of equine strongylids. Bioactive forages like sainfoin (Onobrychis viciifolia) could contribute to reducing drug use, with the first hints of in vitro activity against cyathostomin free-living stages observed in the past. We analysed the effect of a sainfoin-rich diet on cyathostomin population and the efficacy of oral ivermectin treatment. Two groups of 10 naturally infected horses were enrolled in a 78-day experimental trial. Following a 1-week adaptation period, they were either fed with dehydrated sainfoin pellets (70% of their diet dry matter) or with alfalfa pellets (control group) for 21-days. No difference was found between the average fecal egg counts (FECs) of the two groups, but a significantly lower increase in larval devel- opment rate was observed for the sainfoin group, at the end of the trial. Quantification of cyathostomin species abundances with an ITS-2-based metabarcoding approach revealed that the sainfoin diet did not affect the nemabiome structure compared to the control diet. Following oral ivermectin treatment of all horses on day 21, the drug concentration was lower in horses fed with sainfoin, and cyathostomin eggs reappeared earlier in that group. Our results demonstrated that short-term consumption of a sainfoin-rich diet does not decrease cyathostomin FEC but seems to slightly reduce larval development. Consumption of dehydrated sainfoin pellets also negatively affected ivermectin pharmacokinetics, underscoring the need to monitor horse feeding regimes when assessing ivermectin efficacy in the field. [ABSTRACT FROM AUTHOR]

Background: Next-generation sequencing (NGS) has provided an alternative strategy to study the composition of nematode communities with increased resolution and sensitivity. However, the handling and processing of gigabytes worth of amplicon sequence data produced by an NGS platform is still a major hurdle, limiting the use and adoption of faster and more convenient analysis software. Methods: In total 32 paired, fecal samples from Swedish sheep flocks were cultured and the larvae subsequently harvested subjected to internal transcribed spacer 2 (ITS2) amplicon sequencing using the PacBio platform. Samples were analyzed with three different bioinformatic pipelines, i.e. the DADA2, Mothur and SCATA pipelines, to determine species composition and richness. Results: For the the major species tested in this study (Haemonchus contortus, Teladorsagia circumcinta and Trichostrongylus colubriformis) neither relative abundances nor species diversity differed significantly between the three pipelines, effectively showing that all three analysis pipelines, although different in their approaches, yield nearly identical outcomes. In addition, the samples analyzed here had especially high frequencies of H. contortus (90–95% across the three pipelines) both before and after sample treatment, followed by T. circumcinta (3.5–4%). This shows that H. contortus is the parasite of primary importance in contemporary Swedish sheep farms struggling with anthelmintic resistance. Finally, although on average a significant reduction in egg counts was achieved post-treatment, no significant shifts in major species relative frequencies occurred, indicating highly rigid community structures at sheep farms where anthelmintic resistance has been reported. Conclusions: The findings presented here further contribute to the development and application of NGS technology to study nemabiome compositions in sheep, in addition to expanding our understanding about the most recent changes in parasite species abundances from Swedish sheep farms struggling with anthelmintic resistance. [ABSTRACT FROM AUTHOR]

[Display omitted] • Metabarcoded sequencing was used to determined species composition (nemabiome) of gastrointestinal nematode (GIN) larvae. • Deep amplicon sequencing determined proportions of benzimidazole (BZ)-resistant SNPs in Teladorsagia circumcincta. • Nemabiomes varied with time and between ewes and lambs, possibly associated with weather. • The F200Y BZ resistance mutation was close to genetic fixation in T. circumcincta. • There was no genetic evidence of reversion to BZ anthelmintic susceptibility. Anthelmintic resistance threatens the sustainability of sheep production globally. Advice regarding strategies to reduce the development of anthelmintic resistance incorporates the outcomes of modelling exercises. Further understanding of gastrointestinal nematode species diversity, and population dynamics and genetics (which may vary between species) is required to refine these models; and field studies combining faecal egg outputs, species composition and resistance genetics are needed to calibrate them. In this study, faecal samples were taken from ewes and lambs on a commercial farm in south-eastern Scotland at approximately 3 t-4 week intervals between spring and autumn over a period of 4 years. Faecal egg counts were performed on these samples, and L 3 were collected from pooled coprocultures. Deep amplicon sequencing was used to determine both the species composition of these L 3 and the proportions of benzimidazole-resistant single nucleotide polymorphisms in the isotype-1 β-tubulin locus of the predominant species, Teladorsagia circumcincta L 3. Despite consistent management throughout the study, the results show variation in gastrointestinal nematode species composition with time and between age groups, that was potentially associated with weather conditions. The F200Y benzimidazole resistance mutation is close to genetic fixation in the T. circumcincta population on this farm. There was no evidence of variation in isotype-1 β-tubulin single nucleotide polymorphisms frequency between age groups, and no genetic evidence of reversion to benzimidazole susceptibility, despite targeted benzimidazole usage. This study highlights the need to include speciation when investigating gastrointestinal nematode epidemiology and anthelmintic resistance, and serves as an example of how genetic data may be analysed alongside species diversity and faecal egg counts, when markers for other anthelmintic classes are identified. [ABSTRACT FROM AUTHOR]

• Horses are ubiquitously infected by a large diversity of gastro-intestinal parasitic strongyles. • We developed a new ITS2 DNA metabarcoding 'nemabiome' assay to characterise mixed strongyle infections in horses. • The assay differentiates most equine strongyle species with high confidence. • The assay is highly repeatable. • The assay provides reliable information on the relative representation of species in a sample. Horses are ubiquitously infected by a diversity of gastro-intestinal parasitic helminths. Of particular importance are nematodes of the family Strongylidae, which can significantly impact horse health and performance. However, knowledge about equine strongyles remains limited due to our inability to identify most species non-invasively using traditional morphological techniques. We developed a new internal transcribed spacer 2 (ITS2) DNA metabarcoding 'nemabiome' assay to characterise mixed strongyle infections in horses and assessed its performance by applying it to pools of infective larvae from fecal samples from an experimental herd in Kentucky, USA and two feral horse populations from Sable Island and Alberta, Canada. In addition to reporting the detection of 33 different species with high confidence, we illustrate the assay's repeatability by comparing results generated from aliquots from the same fecal samples and from individual horses sampled repeatedly over multiple days or months. We also validate the quantitative potential of the assay by demonstrating that the proportion of amplicon reads assigned to different species scales linearly with the number of larvae present. This new tool significantly improves equine strongyle diagnostics, presenting opportunities for research on species-specific anthelmintic resistance and the causes and consequences of variation in mixed infections. [ABSTRACT FROM AUTHOR]

Abstract Background Many trichostrongylid nematode species are reported to infect bison, some of which are major causes of disase and production loss in North American bison herds. However, there is little information on the species distribution and relative abundance of these parasites in either commercial or conservation herds. This is largely because trichostrongylid nematode species cannot be distinguished by visual microscopic examination of eggs present in feces. Consequently, we have applied ITS2 rDNA nemabiome metabarcoding to describe the trichostrongyle parasite species diversity in 58 bison production groups derived from 38 commercial North American plains bison (Bison bison bison) herds from across western Canada, and two bison conservation herds located in Elk Island National Park (EINP) [plains bison and wood bison (Bison bison athabascae)] and one in Grasslands National Park (GNP) (plains bison). Results We report much higher infection intensities and parasite species diversity in commercial bison herds than previously reported in beef cattle herds grazing similar latitudes. Predominant trichostrongyle parasite species in western Canadian commercial bison herds are those commonly associated with Canadian cattle, with Ostertagia ostertagi being the most abundant followed by Cooperia oncophora. Combined with high fecal egg counts in many herds, this is consistent with significant clinical and production-limiting gastrointestinal parasitism in western Canadian bison herds. However, Haemonchus placei was the most abundant species in five of the production groups. This is both surprising and important, as this highly pathogenic blood-feeding parasite has not been reported at such abundance, in any livestock species, at such northerly latitudes. The presence of Trichostrongylus axei as the most abundant parasite in four herds is also unusual, relative to cattle. There were striking differences in parasite communities between the EINP and commercial bison herds. Most notably, Orloffia bisonis was the predominant species in the wood bison herd despite being found at only low levels in all other herds surveyed. Conclusions This study represents the most comprehensive description of parasite communities in North American bison to date and illustrates the power of deep amplicon sequencing as a tool to study species diversity in gastrointestinal nematode communities.

Understanding how ecology and phylogeny shape parasite communities can inform parasite control and wildlife conservation initiatives while contributing to the study of host species evolution.We tested the relative strengths of phylogeny and ecology in driving parasite community structure in a host whose ecology diverges significantly from that of its closest phylogenetic relatives.We characterized the gastrointestinal (GI) parasite community of wild geladas Theropithecus gelada, primates that are closely related to baboons but specialized to graminovory in the Ethiopian Highlands.Geladas exhibited very constrained GI parasite communities: only two genera (Oesophagostomum and Trichostrongylus) were identified across 305 samples. This is far below the diversity reported for baboons (Papio spp.) and at the low end of the range of domestic grazers (e.g. Bos taurus, Ovis aries) inhabiting the same region and ecological niche.Using deep amplicon sequencing, we identified 15 amplicon sequence variants (ASVs) within the two genera, seven of which matched to Oesophagostomum sp., seven to Trichostrongylus sp., and one to T. vitrinus.Population was an important predictor of ASV richness. Geladas in the most ecologically disturbed area of the national park exhibited approximately four times higher ASV richness than geladas at a less disturbed location within the park.In this system, ecology was a stronger predictor of parasite community structure than was phylogeny, with geladas sharing more elements of their parasite communities with other grazers in the same area than with closely related sister taxa. A free Plain Language Summary can be found within the Supporting Information of this article. [ABSTRACT FROM AUTHOR]

As genomic research continues to improve our understanding of the genetics of anthelmintic drug resistance, the revolution in DNA sequencing technologies will provide increasing opportunities for large-scale surveillance for the emergence of drug resistance. In most countries, parasite control in cattle and bison has mainly depended on pour-on macrocyclic lactone formulations resulting in widespread ivermectin resistance. Consequently, there is an increased interest in using benzimidazole drugs which have been used comparatively little in cattle and bison in recent years. This situation, together with our understanding of benzimidazole resistance genetics, provides a practical opportunity to use deep-amplicon sequencing to assess the risk of drug resistance emergence. In this paper, we use deep-amplicon sequencing to scan for those mutations in the isotype-1 β-tubulin gene previously associated with benzimidazole resistance in many trichostrongylid nematode species. We found that several of these mutations occur at low frequency in many cattle and bison parasite populations in North America, suggesting increased use of benzimidazole drugs in cattle has the potential to result in widespread emergence of resistance in multiple parasite species. This work illustrates a post-genomic approach to large-scale surveillance of early emergence of anthelmintic resistance in the field. [ABSTRACT FROM AUTHOR]

Extensive farming systems form an integral part of sheep production systems across Europe. However, with innate production handicaps, declining sheep numbers and narrow economic margins, production is becoming increasingly challenging threatening the future sustainability of the industry. Gastrointestinal nematodes (GINs) are a significant cause of production losses to the global sheep industry, with well-established resistance to the major anthelmintic groups. Traditionally, extensive farming systems are not thought to have a significant parasite challenge compared with intensive farms, but there is a need to identify the scale and importance of GINs on extensive farms to inform the need for sustainable control strategies. In this study, a questionnaire of extensive farmers (n=34) was conducted and parasitological data were collected from nine study farms to investigate the perceived versus actual GIN and anthelmintic resistance challenge faced by extensive farms. The results showed a production-limiting challenge on most farms, with a higher GIN challenge observed on improved pastures. Furthermore, over half of the extensive farmers perceived anthelmintic resistance to be a greater problem for intensive farmers, with only 20% of respondents reporting known anthelmintic resistance. However, all study farms had evidence of resistance to at least one group of anthelmintics. Consequently, this study has demonstrated that despite the traditional perception of parasitism on extensive farms, there is a need to increasingly consider its impact and take a proactive approach to sustainable control, with solutions tailored to their unique management. • Few surveyed extensive farmers perceived an anthelmintic resistance issue. • All study farms demonstrated evidence of anthelmintic resistance. • Improved grazing tended to have a higher parasite challenge than rough grazing. • Farm management differences will need to be considered for future parasite control. [ABSTRACT FROM AUTHOR]

Our understanding of anthelmintic resistance in the gastrointestinal nematodes of Australian cattle relies exclusively on small-scale phenotypic reports utilising traditional faecal egg count reduction tests. This approach is not readily scalable to establish the national prevalence of resistance, nor is it conducive of routine longitudinal surveillance for the emergence of resistance in its early stages. This study introduces the benefits of applying mixed amplicon metabarcoding longitudinally for timely and cost-efficient molecular surveillance of multiple anthelmintic resistance mutations, as they emerge on farms. Using opportunistically collected faecal samples from a cattle herd in central west New South Wales (2019–2023), we detected the early emergence of Haemonchus spp. levamisole-resistant S168T shortly after levamisole introduction, while benzimidazole-resistant allele frequencies remained constant. Additionally, we observed the possible spill-over of resistant Haemonchus contortus from sheep, along with variations in faecal burdens and species diversity influenced by climate stochasticity and host immunity. This study emphasises the power of molecular diagnostics for farm-level anthelmintic resistance management, providing essential evidence to support its integration into routine surveillance programmes. [Display omitted] • Levamisole-resistant S168T detected shortly after the introduction of levamisole. • Benzimidazole-resistant allele frequencies remained constant throughout the study. • Resistance was rare in specialist cattle nematodes but high in Haemonchus contortus. • Nematode faecal burdens and diversity varied according to climate and host immunity. [ABSTRACT FROM AUTHOR]

A relatively new method to study the species richness and diversity of nematode parasites in grazing animals is to perform deep sequencing on composite samples containing a mixture of parasites. In this work, we compared species composition of strongyles in two groups of horses as a function of egg count and age, based on a DNA barcoding approach. Faecal egg counts and larval cultures were obtained from nearly 300 horses, i.e., domestic horses (n = 167) and trotters (n = 130) sampled nationwide. The second internal transcribed spacer region (ITS2) of strongyle nematodes in the larval cultures was first amplified using barcoded universal primers and then sequenced on the PacBio platform. Subsequently, bioinformatic sequence analysis was performed using SCATA to assign operational taxonomic units (OTU). Finally, species occurrence and composition were assessed using R. ITS2 sequences were found in the majority (89%) of larval samples. Sequencing yielded an average of 140 (26 to 503) reads per sample. The OTUs were assigned to 28 different taxa, of which all but three could be identified as species. The average relative abundance of the seven most abundant species (all Cyathostominae) accounted for 87% of the combined data set. The three species with the highest prevalence in both horse groups were Cyathostomum catinatum, Cylicocyclus nassatus and Cylicostephanus calicatus , and they were frequently found in different combinations with other species regardless of horse group. Interestingly, this result is largely consistent with a previous Swedish study based on morphological analysis of adult worms. In addition, two migratory strongylids (Strongylus vulgaris and S. edentatus) occurred in few domestic horses and trotters. Except for C. minutus and C. nassatus , which decreased with age, and C. catinatum and S. vulgaris , which increased, no specific trends were observed with respect to horse age. Taken together, these results are broadly consistent with data obtained before the introduction of selective targeted treatment in Sweden in 2007. All in all, our results suggest that this treatment strategy has not led to a significant change in strongyle nematode community structure in Swedish horses. The study also confirms that nemabiome analysis in combination with diversity index analysis is an objective method to study strongyle communities in horses. • 28 unique nematode parasite taxa found in 263 Swedish domestic horses and trotters. • The seven most common species were responsible for 87% of the relative abundance. • The three most common species were C. catinatum, C. nassatus, and C. calicatus. • The relative abundance of four species, including S. vulgaris, increased with age. • No influence of selective treatment strategies on strongyle communities was observed. [ABSTRACT FROM AUTHOR]

Information is scarce regarding the epidemiology of gastrointestinal nematodes in Canadian dairy heifers. The objectives of this study were to estimate the prevalence and fecal egg counts of gastrointestinal nematodes in dairy heifers, and using a novel deep-amplicon sequencing approach, to identify the predominant gastrointestinal nematode species in Canadian dairy replacement heifers. Fresh environmental fecal samples (n = 2,369) were collected from replacement heifers on 306 dairy farms across western Canada, Ontario, Québec, and Atlantic Canada. Eggs per gram of feces (EPG) were determined using a modified Wisconsin double-centrifugation sugar flotation technique. Predominant nematode species at the farm level were identified by deep-amplicon nemabiome sequencing of the internal transcribed spacer-2 rDNA locus of nematode third-stage larvae. Generalized estimating equations were used to estimate predicted parasite prevalence and mean EPG in all heifers and by province, allowing for clustering within herds. Individual heifer egg counts ranged from 0 to 141 EPG (median: 0 EPG; interquartile range: 0 to 71 EPG). Gastrointestinal nematodes were detected in 20.9% (95% confidence interval: 17.2 to 24.6%) of heifers, and the predicted mean strongylid EPG accounting for clustering on farms was 1.1 (95% confidence interval: 0.6 to 1.6). The predominant parasite species were Cooperia oncophora and Ostertagia ostertagi. This is the first study in Canada to use a combination of deep-amplicon nemabiome sequencing and a traditional egg count method to describe the epidemiology of gastrointestinal nematodes in dairy heifers. [ABSTRACT FROM AUTHOR]

The study presents the results of a cross-sectional survey to describe the epidemiology of ascarid and strongylid nematodes in horses, the impact of diverse climatic conditions on parasite diversity and the levels of faecal egg shedding in different age groups of managed Thoroughbred horses. Individual faecal samples (n = 1377) collected from 62 Thoroughbred farms across four climatic zones in Australia were analysed using the modified McMaster technique for faecal egg counts (FECs) and strongylid nematodes were identified utilising PCR-directed next-generation sequencing (NGS) of the second internal transcribed spacer of the nuclear ribosomal DNA (ITS-2). Across all age groups, the prevalence of ascarid and strongylid nematodes was 12% (95% confidence interval 10–14%) and 72% (70–74%), respectively. Based on strongylid FECs, yearlings had the highest prevalence (89%) followed by weanlings (83%), foals (79%), wet mares (61%), dry mares (59%) and stallions (54%). However, for Parascaris spp., foals had the highest prevalence (46%) followed by weanlings (32%) and yearlings (13%). The highest mean FECs for Parascaris spp. were observed in foals (418 eggs per gram [EPG] of faeces) while those for strongylids were in yearlings (1002 EPG). Of the adult horses (mares and stallions), 67% (489 of 729) and 11% (77 of 729) were low (i.e., ≤250 EPG) and moderate (i.e., 251–500 EPG) strongylid egg-shedders, respectively. Strongylid egg shedding varied across climatic zones, with the highest mean FECs in the summer rainfall (723 EPG) followed by non-seasonal rainfall (629 EPG), winter rainfall (613 EPG), and Mediterranean (606 EPG) rainfall zones. Twenty-three nematode species were detected using NGS, with Cylicostephanus longibursatus (28%), Cylicocyclus nassatus (23%) and Coronocyclus coronatus (23%), being the most abundant species. Three species of Strongylus (i.e., S. vulgaris, S. equinus and S. edentatus) were also detected. The nemabiome composition, species richness and relative abundance varied within horse age and between climatic zones. These empirical findings provide a comprehensive understanding of the prevalence of parasites within horse populations and the multifaceted factors that influence their occurrence, thereby allowing for the formulation of tailored strategies aimed at parasite control in domestic horses. [Display omitted] • A cross-sectional study of 62 Thoroughbred farms revealed a high prevalence of ascarid and strongylid nematodes. • Majority of the adult horses were low strongylid egg shedders (i.e., < 250 eggs per gram). • Horse age and diverse climatic conditions impacted the parasite diversity and egg shedding patterns. • Cylicostephanus longibursatus, Coronocyclus coronatus and Cylicocyclus nassatus were the most abundant species. • Study provided insights for tailoring parasite control strategies in horses. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Deep amplicon sequencing is a powerful new tool for drug resistance single nucleotide polymorphism (SNP) screening. • Deep amplicon sequencing is validated by pyrosequence genotyping of individual nematode larvae. • There is an high frequency of benzimidazole resistance SNPs in many nematode species on UK sheep farms. • Observed resistance SNP frequencies differ greatly between species under the same drug selection. Abstract Parasitic gastrointestinal nematodes contribute to significant human morbidity and cause billions of dollars per year in lost agricultural production. Control is dependent on the use of anthelmintic drugs which, in the case of livestock parasites, is severely compromised by the widespread development of drug resistance. There are now concerns regarding the emergence of anthelmintic resistance in parasitic nematodes of humans in response to the selection pressure resulting from mass drug administration programs. Consequently, there is an urgent need for sensitive, scalable and accurate diagnostic tools to detect the emergence of anthelmintic resistance. Detecting and measuring the frequency of resistance-associated mutations in parasite populations has the potential to provide sensitive and quantitative assessment of resistance emergence from an early stage. Here, we describe the development and validation of deep amplicon sequencing as a powerful new approach to detect and quantify the frequency of single nucleotide polymorphisms associated with benzimidazole resistance. We have used parasite communities in sheep to undertake a proof-of-concept study of this approach. Sheep provide an excellent host system, as there are multiple co-infecting trichostrongylid nematode species, each likely with a varying prevalence of benzimidazole resistance. We demonstrate that the approach provides an accurate measure of resistance allele frequencies, and can reliably detect resistance alleles down to a frequency of 0.1%, making it particularly valuable for screening mutations in the early stages of resistance. We illustrate the power of the technique by screening UK sheep flocks for benzimidazole resistance-associated single nucleotide polymorphisms at three different codons of the β-tubulin gene in seven different parasite species from 164 populations (95 from ewes and 69 from lambs) in a single MiSeq sequencing run. This approach provides a powerful new tool to screen for the emergence of anthelmintic resistance mutations in parasitic nematode populations of both animals and humans. [ABSTRACT FROM AUTHOR]

Next-generation deep amplicon sequencing, or metabarcoding, has revolutionized the study of microbial communities in humans, animals and the environment. However, such approaches have yet to be applied to parasitic helminth communities. We recently described the first example of such a method – nemabiome sequencing – based on deep-amplicon sequencing of internal transcribed spacer 2 (ITS-2) rDNA, and validated its ability to quantitatively assess the species composition of cattle gastro-intestinal nematode (GIN) communities. Here, we present the first application of this approach to explore GIN species diversity and the impact of anthelmintic drug treatments. First, we investigated GIN species diversity in cow-calf beef cattle herds in several different regions, using coproculture derived L3s. A screen of 50 Canadian beef herds revealed parasite species diversity to be low overall. The majority of parasite communities were comprised of just two species; Ostertagia ostertagi and Cooperia oncophora . Cooperia punctata was present at much lower levels overall, but nevertheless comprised a substantive part of the parasite community of several herds in eastern Canada. In contrast, nemabiome sequencing revealed higher GIN species diversity in beef calves sampled from central/south-eastern USA and Sao Paulo State, Brazil. In these regions C. punctata predominated in most herds with Haemonchus placei predominating in a few cases. Ostertagia ostertagi and C. oncophora were relatively minor species in these regions in contrast to the Canadian herds. We also examined the impact of routine macrocyclic lactone pour-on treatments on GIN communities in the Canadian beef herds. Low treatment effectiveness was observed in many cases, and nemabiome sequencing revealed an overall increase in the proportion of Cooperia spp. relative to O. ostertagi post-treatment. This work demonstrates the power of nemabiome metabarcoding to provide a detailed picture of GIN parasite community structure in large sample sets and illustrates its potential use in research, diagnostics and surveillance. [ABSTRACT FROM AUTHOR]

Gastrointestinal nematodes (GIN) are amongst the most important pathogens of grazing ruminants worldwide, resulting in negative impacts on cattle health and production. The dynamics of infection are driven in large part by the influence of climate and weather on free-living stages on pasture, and computer models have been developed to predict infective larval abundance and guide management strategies. Significant uncertainties around key model parameters limits effective application of these models to GIN in cattle, however, and these parameters are difficult to estimate in natural populations of mixed GIN species. In this paper, recent advances in molecular biology, specifically ITS-2 rDNA 'nemabiome' metabarcoding, are synthesised with a modern population dynamic model, GLOWORM-FL, to overcome this limitation. Experiments under controlled conditions were used to estimate rainfall constraints on migration of infective L3 larvae out of faeces, and their survival in faeces and soil across a temperature gradient, with nemabiome metabarcoding data permitting species-specific estimates for Ostertagia ostertagi and Cooperia oncophora in mixed natural populations. Results showed that L3 of both species survived well in faeces and soil between 0 and 30 °C, and required at least 5 mm of rainfall daily to migrate out of faeces, with the proportion migrating increasing with the amount of rainfall. These estimates were applied within the model using weather and grazing data and use to predict patterns of larval availability on pasture on three commercial beef farms in western Canada. The model performed well overall in predicting the observed seasonal patterns but some discrepancies were evident which should guide further iterative improvements in model development and field methods. The model was also applied to illustrate its use in exploring differences in predicted seasonal transmission patterns in different regions. Such predictive modelling can help inform evidence-based parasite control strategies which are increasingly needed due climate change and drug resistance. The work presented here also illustrates the added value of combining molecular biology and population dynamics to advance predictive understanding of parasite infections. • Nemabiome sequencing was combined with modelling nematode populations. • Models were adapted for Ostertagia ostertagi and Cooperia oncophora in N.America. • Updated life-history parameters were defined based on in vitro observations. • The refined model can effectively predict the seasonal patterns of cattle GIN. [ABSTRACT FROM AUTHOR]

• Sequencing of nematode L 1 versus L 3 may reduce bias due to culture conditions. • Much higher proportions of small ruminant nematode eggs are recovered as L 1 than L 3. • Handling of small ruminant feces prior to culture may influence speciation results. Mixed gastrointestinal nematode (GIN) infections are a common and significant cause of financial loss for small ruminant producers. Morphologic examination of third-stage larvae (L 3) can be used to identify species composition in feces but has limitations due to the requirement for specialized expertise and the extensive time (8–15 d depending on method used) and labour involved. Moreover, differential development and survival of larvae during coproculture to the third stage often occurs. Deep amplicon sequencing of the ITS-2 rDNA locus of first-stage larvae (L 1) allows for higher throughput with reduced specialist labour and reduces the risk of misidentification. Harvesting of L 1 soon after hatching is also faster and further reduces labour as well as biases that can occur due to differential larval development and survival. This study compares the results of morphologic examination of L 3 with those of ITS-2 rDNA deep amplicon sequencing of L 1 from a set of pooled fecal samples. The proportions of eggs that were successfully recovered as larvae following culture to L 3 and L 1 were also compared. Larval recovery rate was significantly lower from L 3 cultures than from L 1 cultures (p < 0.001); eggs were 238.7 times less likely to develop to L 3 than to L 1 (95 % confidence interval for odds ratio 80.0–712.0). Significantly lower proportions of Teladorsagia circumcincta (odds ratio = 3.1, p = 0.008) and higher proportions of Trichostrongylus spp. (p = 0.009) were identified using morphologic examination of L 3 compared with deep amplicon sequencing of L 1 on the same samples. This is consistent with previous reports of differential survival of these species in L 3 cultures. These results indicate that deep amplicon sequencing of L 1 may reduce bias introduced by differential GIN survival to L 3 in small ruminants. [ABSTRACT FROM AUTHOR]

• One third of the samples analyzed were positive for Clade V nematode DNA. • Ostertagia was found in 90 % of the sequenced samples. • Wild ruminants can act as reservoirs for maintaining infections in production herds. Gastrointestinal nematode (GIN) infections in cattle cause appetite suppression which leads to poor feed conversion, reduced weight gain and reduced milk production. Overuse and exclusive reliance on anthelmintic drugs has resulted in widespread resistance in many parasitic nematode species infecting livestock making control increasingly difficult. Wild ruminants are competent hosts of a number of nematode species that typically infect and are best adapted for cattle, sheep, and goats. Thus, the potential exists for wild ruminants to act as reservoirs in the translocation of domestic GIN, including those carrying anthelmintic resistance mutations as well as susceptible genotypes. The potential for parasite exchange is heightened by interfaces or ecotones between managed and wild rangelands, and by perturbations linked to climate warming that can increasingly alter the distributions of wild ungulates and their interactions with domestic and free-ranging ruminants. To investigate the extent to which wild ruminants harbour parasites capable of infecting domestic ruminants we first performed an epidemiological study of feces from wildlife hosts that spanned 16 states and included white-tailed deer (85 % of the samples), pronghorn, elk, mule deer, bighorn sheep, moose, cattle, and caribou across the United States. All samples were cultured to third stage larvae and nematode DNA was isolated and PCR amplified. Among the 548 wild ruminant samples received, 33 % (181 samples) were positive for nematode DNA, among which half (84 samples) contained DNA from GIN species commonly found in cattle. DNA from cattle GIN species was detected in 46 % of samples from the Northeast, 42 % from the Southeast, 10 % from the Midwest, 0 % from the Southwest and 11 % from the West. Deep amplicon sequencing of the ITS-2 rDNA indicated that Ostertagia and Trichostrongylus were present in 90 % and 69 % of the nematode DNA positive samples, respectively, whereas Haemonchus, Cooperia and Oesophagostomum were present in 26 %, 2 % and 10 % of the samples, respectively. These data clearly show that wild ruminants commonly harbour multiple parasite species whose primary hosts are domestic cattle, and suggest that further work is warranted to investigate their specific roles in the management of anthelmintic resistance. [ABSTRACT FROM AUTHOR]